The Chaperone Activity and Substrate Spectrum of Human Small Heat Shock Proteins

Abstract

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that suppress the unspecific aggregation of miscellaneous proteins. Multicellular organisms contain a large number of different sHsps, raising questions as to whether they function redundantly or are specialized in terms of substrates and mechanism. To gain insight into this issue, we undertook a comparative analysis of the eight major human sHsps on the aggregation of both model proteins and cytosolic lysates under standardized conditions. We discovered that sHsps, which form large oligomers (HspB1/Hsp27, HspB3, HspB4/αA-crystallin, and HspB5/αB-crystallin) are promiscuous chaperones, whereas the chaperone activity of the other sHsps is more substrate-dependent. However, all human sHsps analyzed except HspB7 suppressed the aggregation of cytosolic proteins of HEK293 cells. We identified ∼1100 heat-sensitive HEK293 proteins, 12% of which could be isolated in complexes with sHsps. Analysis of their biochemical properties revealed that most of the sHsp substrates have a molecular mass from 50 to 100 kDa and a slightly acidic pI (5.4-6.8). The potency of the sHsps to suppress aggregation of model substrates is correlated with their ability to form stable substrate complexes; especially HspB1 and HspB5, but also B3, bind tightly to a variety of proteins, whereas fewer substrates were detected in complex with the other sHsps, although these were also efficient in preventing the aggregation of cytosolic proteins.

Keywords: cataract; molecular chaperone; protein aggregation; protein stability; small heat shock protein (sHsp); substrate proteins; α crystallin.

FIGURE 1.

The effect of human sHsps (HspB1-HspB8) on the aggregation of six different model substrates. Left panels, aggregation of model substrates was monitored in the presence of increasing concentrations of different sHsps. The levels of aggregation (apparent absorbance at 360 nm) at the end of the incubation period were normalized to the absorbance signal of spontaneous aggregation (without sHsp) and plotted against the sHsp concentration. Here and in all other figures the color code is as shown in the right bottom corner: black, HspB1; magenta, HspB2; orange, HspB3; blue, HspB4; cyan, HspB5; red, HspB6; brown, HspB7; green, HspB8; gray, without sHsp. Right panels, aggregation kinetics of six model substrates in the presence of different human sHsps at one selected ratio indicated by the red line in the graphs on the left side. The model substrates were incubated alone or with different sHsps at 45 °C (MDH, GAPDH, rhodanese, and CS), 42 °C (ADH), or 37 °C (insulin), and aggregation kinetics were followed by monitoring the apparent optical density at 360 nm. The mean of at least three independent replicates ± S.D. is shown in all graphs. a.u., arbitrary units.

FIGURE 2.

Temperature-induced aggregation of HeLa, HEK293, and MCF7 cell lysates in the absence and presence of human sHsps. A, 0.8 mg/ml lysate of the corresponding cell line (indicated on the top) was incubated at 4 °C (4) or 45 °C (45) for 90 min in PBS, 1 mm DTT. After incubation, soluble (S) and insoluble (pellet, P) fractions were separated by centrifugation and analyzed by SDS-PAGE. B, samples containing HEK293 lysate were incubated for 90 min at 45 °C either without sHsps (first lanes, 0) or with different amounts (concentration in μm as indicated) of HspB1-HspB8. Bands corresponding to the sHsp added are indicated by arrows on the right side of each gel. After heat shock, the soluble and insoluble proteins were separated by centrifugation, and the latter fraction was analyzed by SDS-PAGE. Positions of molecular weight markers are indicated in kDa. C, suppression of HEK293 and HeLa cell lysate protein aggregation by sHsps. The total amount of insoluble proteins in the samples was quantified by densitometry of the corresponding lanes shown in B for HEK293 lysates (gels for HeLa lysates are not shown) and normalized to the total amount of insoluble proteins in samples without sHsp (lanes 0 in B, was set to 1). In addition to the sHsps, GFP was used as a control protein, which does not affect the cell lysate aggregation at the respective concentrations (violet curves). The mean of three independent experiments ± S.D. is shown. a.u., arbitrary units.

FIGURE 3.

Substrate spectra of human small heat shock proteins. A, the total number of interactors for HspB1-HspB7 detected in pulldown experiments; the first bar (dark green) shows the number of total unique hits detected. B, overlap in potential substrates of HspB1, HspB3, and HspB5. C–E, statistical analysis (box plot) of molecular mass (C), pI (D), and hydrophobicity (GRAVY index) (E) distributions of detected sHsps substrates in comparison to a heat-sensitive fraction of HEK293 lysate (HEK HS) and to the total human proteome. F, abundance distributions of proteins from the HEK293 cell line (HEK total) (40), from HEK293 heat-sensitive fraction (HEK HS), and all detected sHsp substrates. G, overrepresentation of GO categories for substrate proteins of HspB1-HspB5. The analysis was performed with the PANTHER online tool using default settings and the total human proteome as the reference set. Only classes with p < 0.05 are shown.

FIGURE 4.

Overview of the chaperone properties of human sHsps. Except for the last column, the numbers are IC₅₀ values that represent the ratio of sHsp to substrate required for half-maximum aggregation suppression. The last column indicates the number of sHsp interactors detected in pulldown experiments. Colors correspond to the relative activity in the given aggregation assay; dark blue, sHsp is highly active; light blue, sHsp is inactive or slightly active. n.a. indicates that the respective sHsp is not active or co-aggregates with the substrate. n.d., coimmunoprecipitation was not done for HspB8. * indicates sHsps that were proteolyzed during the cell lysate assays.